1. Field of the Invention
Affinity chromatography has become a valuable tool for separating biological materials, for example, biologically active molecules such as small ligands, proteins, nucleic acids, enzymes, etc. In affinity chromatography, a substrate is immobilized on a granular support. By utilizing a column of said granular, immobilized substrate, materials having affinity, structure selectivity, or binding specificity for the compounds bonded to the stationary phase can be separated from other materials in an aqueous mixture thereof.
Typically, complex polysaccharide granules such as agarose beads, etc. are employed in affinity chromatography. The material of the granules may inherently have the desired sorption specificity or a suitable ligand may be bonded, often through so-called spacer-arms, to the granules by a variety of methods. Components in an ambient solution which have a high binding affinity for the materials of the granules or for the ligand will be preferentially bound to the granules. The bound components may be subsequently removed from the granules by contacting the latter with reagents which reduce the binding of the component for the material of the granules or for the ligand. Typically such desorption is accomplished by a substantial change in pH or ionic strength or both. Alternatively the sorbed component may be desorbed by another ligate which competes for the binding sites. Often chaotropic agents are used which, apparently, by altering the secondary structure of the sorbed component effect desorption.
Examples of highly specific ligands are:
Lectin absorbents for binding glycoproteins, glycolipids, polysaccharides, and related substances; PA1 Protein A from Staphylococcus aureus for binding many immunoglobulins; PA1 Cibacron.RTM. Blue F3G-A for binding albumin, interferons, growth factors, kinases and dehydrogenases; PA1 Monoclonal antibodies for binding biospecific antigens; PA1 Biospecific antigens for binding monoclonal antibodies; and PA1 Hydrophobic groups (e.g. aliphatic or aromatic moieties) for binding proteins having hydrophobic regions.
Desorption of tightly bound, high molecular weight ligates is generally a rather slow process, particularly in porous granules or beads, and in the presence of substantial changes in pH, ionic strength or high concentrations of chaotropic reagents may result in alterations in biological activity of the ligate or loss of biospecificity of the ligands.
It is an object of the present invention to provide processes and apparatuses which enable easy handling of the biospecific substrate and the comparatively rapid and economic recovery of bioactive ligates under comparatively gentle desorption conditions, thereby preserving a substantial fraction of the bio-activity.
These and other objects will become apparent from the following description of the invention.
2. Description of the Prior Art
The prior art is well described in:
(a) "Affinity Chromatography" by W. H. Scouten, John Wiley & Sons, Inc., New York, 1981; PA0 (b) "Affinity Chromatography" by J. Turkova, Elsevier, Amsterdam, Netherlands, 1978; PA0 (c) "Affinity Chromatography, Principles and Methods", Pharmacia Fine Chemicals AB, Uppsala, Sweden; and PA0 (d) "Kirk-Othmer Encyclopedia of Chemical Technology, 3rd Edition, Vol 6, page 35 et. seq.
Typical support materials for affinity chromatography are granular and bead-like gels of agarose, cross-linked agarose, polyacrylamide and dextranepichlorohydrin. They are high cost, soft and compressible which make them unattractive for large scale processes. Rigid supports can be made from porous glass or ceramic beads which are coated to reduce non-specific protein binding. These are technically suited for large scale processes but are even more expensive than gel beads and granules.
To obtain high binding capacities with bead or granular supports it is necessary to use the interior as well as the exterior area of the support, that is, the support must have high porosity for the typically high molecular weight materials sorbed. However, access to such interior surfaces is very slow for high molecular weight sorbates leading to long sorption and desorption times. Since desorption often requires pronounced changes in pH or ionic strength and/or the presence of chaotropic agents, long desorption times can lead to irreversible changes in the secondary structure of the ligates and/or ligands. However, access to such interior surfaces is very slow for high molecular weight sorbates leading to long sorption and desorption times. Since desorption often requires pronounced changes in pH or ionic strength and/or the presence of chaotropic agents, long desorption times can lead to irreversible changes in the secondary structure of the ligates and/or ligands. Polyacrylamide and dextranepichlorohydrin gels with suitable mechanical properties typically have low molecular porosity.
U.S. patent application Ser. No. 675,057 now U.S. Pat. No. 4,584,075, filed Nov. 26, 1984, entitled "Process and Apparatus for Electrically Desorbing Components Selectively Sorbed on Electrolytic Conducting Barriers" and assigned to the same assignee as this patent application, discloses processes and apparatuses for recovering components dispersed in an aqueous solution comprising contacting said solution as a first solution with an electrically conductive barrier which has a high affinity for one of said components. The barrier is subsequently contacted with a second aqueous solution and a direct electric current is passed through said barrier in a direction substantially perpendicular to the barrier and to the flow of solution thereby resulting in the substantial desorption of the sorbed component. Said application does not disclose recovering components sorbed on a body of fibrous objects which have a high affinity for at least one of said components.
U.S. patent application Ser. No. 703,581 now U.S. Pat. No. 4,594,135, filed Feb. 20, 1985, entitled "Process and Apparatus for Electrically Desorbing Components Selectively Sorbed on Granules" (of which the present invention is a Continuation-In-Part and assigned to the same assignee as this patent application) discloses process and apparatus for recovering components sorbed on a body of wet granules which are highly selective for the sorption of said components. The process comprises passing a direct electric current through said body of granules and an ion-permeable, substantially hydraulically impermeable barrier juxtaposed with said body of granules in a direction substantially parallel to the smallest dimension of said barrier, thereby facilitating the desorption of at least one of said selected components from said granules.
All the prior art substrates summarized above suffer from high cost and/or, if they contain high available surface areas, poor mechanical properties. If such substrates have high internal surface areas then undesirably long sorption and desorption times will be experienced for high molecular weight ligates, often leading to substantial denaturation of the ligate and/or ligand.
It is therefore a further objective of this invention to provide substrates having low cost per unit volume and good mechanical properties. It is also an objective to provide apparatuses and methods for using such substrates in affinity separations.
These and other objectives will become clear from the following brief description of the drawings and description of preferred embodiments.